Structure of recombinant Tau40 protein fibrils prepared without enhancers of fibrilization

Gytis Kučinskas^1,2, Dr. Jozef Hritz^1,3

¹Central European Institute of Technology (CEITEC) Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

²National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

³Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

gytis.kucinskas@ceitec.muni.cz, jozef.hritz@ceitec.muni.cz

Neurodegenerative diseases such as Alzheimer’s disease remain a global issue with an increasing number of patients and without effective therapeutical solution. Tau protein fibrils are structured aggregates present in AD patient’s brains. Accumulation of these Tau fibrils lead to neuronal cell death, decrease in brain density and progression of the AD. Under normal physiological conditions Tau binding to microtubules is responsible for stabilization of the microtubule network in axons.1 This stabilization is regulated by posttranslational modifications.2 However, some posttranslational modifications of Tau such as hyperphosphorylation and truncation are widely described as key pathological factors in formation of Tau fibrils with different modification resulting in structurally different filaments.

Recently cryo-EM showed that Tau fibrils isolated from brain of AD patients are present in two distinct fibril formations.3 It was also shown that heparin induced recombinant Tau fibrils formed different types of filaments than filaments isolated from AD brains.4 These results show the importance of origin and type of Tau fibrils while characterizing the fibril structure.

Our study focuses on structure of Tau filaments formed by recombinant full length Tau isoform (Tau40) prepared by fibrilization that does not require any enhancers. This isoform is the longest of all 6 isoforms5 and considered to be the most abundant. We tested several different conditions of fibrilization based on type of buffer, temperature, presence or absence of agitation and additives. Fibrilization rate was quantified using Thioflavin T assay that allowed to detect the formation of fibrils by the increase in fluorescence response. Fibrils were visualized using negative staining, cryo-EM and atomic force microscopy (AFM). Based on our preliminary results, we found that the best fibril growth was obtained with phosphate buffers at 37 degrees with 700 rpm agitation. These fibrils are being further analyzed by cryo-EM with a goal to reconstruct their atomic structure in order to test if our recombinant Tau fibrils prepared without fibrilization enhancers match the fibrils isolated from brains of AD patients.

1. Kellogg, E.H., Hejab, N.M., Poepsel, S., Downing, K.H., DiMaio, F. and Nogales, E., 2018. Near-atomic model of microtubule-tau interactions. Science, 360(6394), pp.1242-1246.

2. Dehmelt, L. and Halpain, S., 2005. The MAP2/Tau family of microtubule-associated proteins. Genome biology, 6(1), pp.1-10.

3. Fitzpatrick, A.W., Falcon, B., He, S., Murzin, A.G., Murshudov, G., Garringer, H.J., Crowther, R.A., Ghetti, B., Goedert, M. and Scheres, S.H., 2017. Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature, 547(7662), pp.185-190.

4. Zhang, W., Falcon, B., Murzin, A.G., Fan, J., Crowther, R.A., Goedert, M. and Scheres, S.H., 2019. Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases. Elife, 8, p.e43584.

5. Goedert, M.G.S.M., Spillantini, M.G., Jakes, R., Rutherford, D. and Crowther, R.A., 1989. Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron, 3(4), pp.519-526.

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic within programme INTER-ACTION (project no. LTAUSA18168). CIISB research infrastructure project LM208127 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CEITEC Cryo-Electron Microscopy and Tomography Core Facility, CEITEC Proteomics Core Facility and CEITEC Nanobiotechnology Core Facility.